Abstract

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (KATP); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 ± 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 ± 1.9%, 11,12-EET; and 8.4 ± 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 ± 2.1%, 11,12-EET; and 9.7 ± 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 ± 4.6%) by the KATP channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 ± 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP ω-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 ± 1.2%, DDMS; and 22.2 ± 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP ω-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP ω-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac KATP channels.

cytochrome P-450 epoxygenase

11,12-epoxyeicosatrienoic acid

14,15-epoxyeicosatrienoic acid

20-hydroxyeicosatetraenoic acid

ischemic preconditioning

adenosine 5′-triphosphate-sensitive potassium channels

recently, the influence of the cytochrome P-450 pathway (CYP) has been investigated in several animal models of ischemia-reperfusion injury by our laboratory and others (3, 4, 10, 12, 13). Initially, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 demonstrated an improvement in functional recovery after 20 min of global ischemia and 40 min of reperfusion. This beneficial action was hypothesized to be the result of an increased production of epoxyeicosatrienoic acids (EETs) because administration of exogenous 11,12-EET also produced a similar cardioprotective effect in control mice. More recently, treatment with several nonselective CYP inhibitors resulted in a reduction in myocardial infarct size (IS) in mice and rats, and the reduction of IS was attributed to the ability of these compounds to prevent CYP-derived oxygen free radical release (2, 3).

We (9, 12) previously found high plasma concentrations of the CYP ω-hydroxylase metabolite, 20-HETE, during ischemia and after reperfusion in the canine heart. We also demonstrated that several CYP ω-hydroxylase isoforms that potentially could produce 20-HETE, such as CYP4A1, CYP4A2, and CYP4F, are present in canine myocardium. Based on these results, we administered several inhibitors of 20-HETE formation or its action, such as miconazole, 17-octadecynoic acid, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), to canine hearts and determined IS. All four compounds markedly inhibited 20-HETE production during ischemia-reperfusion, and all produced marked reductions in myocardial IS (4, 10, 12). These beneficial effects on IS were interpreted as occurring because of a favorable increase in the ratio of the cardioprotective EETs versus 20-HETE in the heart; however, no direct evidence was presented to show that EETs were responsible for these observed effects.

Because the EETs are a group of endogenous compounds that have been shown to be cardioprotective against myocardial stunning, a degree of injury that is much less severe than that which occurs during a myocardial infarction, the present study was performed to determine the effects of two EET regioisomers, 11,12-EET and 14,15-EET, on myocardial infarction and to compare their effects with that of ischemic preconditioning (IPC), another endogenous mediator of cardioprotection. In this regard, we wanted to test the hypothesis that release of endogenous EETs during IPC or during prolonged ischemia contributes to IPC-induced cardioprotection by blocking CYP epoxygenases, the enzymes that are the major sources of EET production, in the heart with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), a selective inhibitor of EET synthesis, during IPC. We also determined whether the effect of DDMS, a selective CYP ω-hydroxylase inhibitor, that produces cardioprotection by tipping the endogenous balance between 20-HETE and the EETs in favor of the EETs could be mediated by enhanced EET synthesis and release in dogs by inhibiting EET synthesis by the simultaneous administration of MS-PPOH, a selective CYP epoxygenase inhibitor, and DDMS, a selective CYP ω-hydroxylase inhibitor.

Finally, several studies have suggested that the EETs are direct activators of sarcolemmal ATP-sensitive potassium (sarcKATP) channels in membrane patches isolated from rat myocardium (6, 7) and have been shown to activate mitochondrial KATP (mitoKATP) channels in mouse hearts (13). Because both of these channels have been shown to be important in mediating cardioprotection produced by numerous mediators in many laboratories, including our own (5), we decided to test the importance of this channel in mediating the cardioprotective effects of the EETs in our canine model of infarction.

General preparation of dogs.

All experiments conducted in this study were performed in accordance with the “Position of the American Heart Association on Research and Animal Use,” adopted by the American Heart Association, and approved by the Biomedical Resource Center and the Institutional Animal Care Committee of the Medical College of Wisconsin. The Medical College of Wisconsin is accredited by the American Association of Laboratory Animal Care.

The protocol for dog preparation and experiments has been previously described in detail (12). Briefly, adult mongrel dogs of either sex, weighing 18 to 25 kg, were fasted overnight, anesthetized with the combination of barbital sodium (200 mg/kg) and pentobarbital sodium (15 mg/kg), and ventilated with room air that was supplemented with 100% oxygen. Body temperature was maintained at 38 ± 1°C with a heating pad. Atelectasis was prevented by maintaining an end-expiratory pressure of 5–7 cmH2O with a trap. Arterial blood pH, Pco2, and Po2 were monitored at selected intervals by an AVL automatic blood-gas system and maintained within normal physiological limits (pH, 7.35 to 7.45; Pco2, 30 to 40 mmHg; and Po2, 85 to 120 mmHg) by adjusting the respiration rate and oxygen flow or by intravenous administration of 1.5% sodium bicarbonate if necessary. A flowmeter (Statham 2202) was used to measure left anterior descending coronary artery (LAD) blood flow. A mechanical occluder was placed distal to the flow probe such that there were no branches between the flow probe and the occluder. Hemodynamics, heart rate, and coronary blood flow were monitored throughout the experiment. The left atrium was cannulated via the appendage for radioactive microsphere injections.

Experimental design.

Dogs were sequentially assigned to 12 groups for different treatments. Typically, eight dogs were used in each group of experiments. At 15 min before the 60-min LAD occlusion period, 11,12-EET (0.128 mg/kg), 14,15-EET (0.128 mg/kg), 14,15-DHET (0.155 mg/kg), DDMS (0.81 mg/kg), or vehicle were administered by intracoronary infusion for 2–3 min as shown in protocol 1 (Fig. 1). For IPC experimental groups, a brief occlusion of 5 min was performed 15 min after the administration of drugs (or vehicle) and the subsequent 60-min LAD occlusion (10 min after IPC) as shown in protocol 2 (Fig. 1). In two other groups, these same doses of EETs were administered by intratrial infusions over 2–3 min at 5 min before reperfusion to determine whether EETs block reperfusion injury. Glibenclamide (1.0 mg/kg iv) was administered 5 min before 11,12-EET (0.128 mg/kg ic). Finally, MS-PPOH, a CYP epoxygenase inhibitor, was administered either alone 20 min before occlusion or concomitantly with IPC or DDMS. In all groups, hemodynamic measurements, blood-gas analyses, and myocardial blood flow measurements were performed at baseline and at 30 min into the 60-min occlusion period. After reperfusion, hemodynamics were measured every hour and myocardial blood flow was determined at the end of the 3-h reperfusion period. At the end of the experiment, the hearts were electrically fibrillated, removed, and prepared for IS determination and regional myocardial blood flow measurement.

IS determination.

IS was determined as previously described (8). Briefly, at the end of the 3-h reperfusion period, the LAD was cannulated. To determine the anatomic area at risk (AAR) and the nonischemic area, 5 ml of Patent blue dye and 5 ml of saline were injected at equal pressure into the left atrium and LAD, respectively. The heart was then immediately fibrillated and removed. The left ventricle (LV) was dissected and sliced into serial transverse sections 6–7 mm in width. The nonstained ischemic area and the blue-stained normal area were separated and incubated with 1% 2,3,5-triphenyltetrazolium chloride (Sigma) in 0.1 mol/l phosphate buffer, pH 7.4 at 37°C for 15 min. After incubation overnight in 10% formaldehyde, the noninfarcted and infarcted tissues within the AAR were separated and determined gravimetrically. IS was expressed as a percentage of the AAR (IS/AAR).

Regional myocardial blood flow.

Regional myocardial blood flow (in ml·min−1·g−1) was measured by the radioactive microsphere technique as previously reported (1). Microspheres were administered at 30 min into the 60-min occlusion period and at the end of 3 h of reperfusion. Transmural blood flow was calculated as the weighted average of the subepicardium, midmyocardium, and subendocardium in each region.

Sample collection and preparation for CYP metabolites of AA determination.

In some experiments, coronary venous plasma (great cardiac vein) samples were collected as previously described (9). Briefly, blood was drawn through an 8-Fr single lumen (100 cm length) catheter. Once the catheter was cleared of residual, stagnant blood, 5 ml of blood was aspirated over a 3- to 5-s period into a chilled sample tube containing heparin (1,000 U/ml) and miconazole (5 × 10−5 mol/l), mixed thoroughly and placed in an ice bucket. Blood samples were collected at different times as indicated in protocols 1 and 2 (Fig. 1). The samples were centrifuged at 3000 g at 0°C for 10 min to separate plasma. Plasma was removed and transferred to a tube, purged with nitrogen gas, and capped and stored at −80°C or extracted.

The internal standards, 1.0 ng each for [2H8]EETs, [2H8]DHETs, and 20-[2H2]HETE, were added to each sample and mixed. Ethanol was then added to a final concentration of 15% and mixed and centrifuged at 1,500 rpm for 3 min. Plasma samples were extracted by SPE as previously described (11).

CYP metabolites of AA in plasma samples were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (Agilent 1100 LC/MSD, SL Model) as previously described (11). Selected ion monitoring in the negative detection mode was used for determination of the CYP metabolites of AA.

Statistical analysis.

All values are expressed as means ± SE. Differences between groups in hemodynamics were compared by using a two-way ANOVA. Differences between groups in tissue blood flows, AAR, and IS were compared by one-way ANOVA, and differences in concentrations of AA metabolites at various times during occlusion and after reperfusion between treatment groups and the control group were compared by using a two-way repeated-measures ANOVA, followed by a Tukey's post hoc test. Differences between groups were considered significant if P < 0.05.

RESULTS

Regional myocardial blood flow.

Transmural blood flows in the nonischemic (left circumflex coronary) and the ischemic (LAD) regions were measured during 60 min of occlusion and after 3 h of reperfusion. There was no significant difference in nonischemic or transmural collateral blood flow (Table 1) between groups, indicating that all groups were subjected to similar degrees of ischemia. Similarly, there were no significant differences in the AAR between groups (data not shown). Thus the two major determinants of IS in the dog, collateral blood flow and the AAR, were not different, so that any changes in IS observed in the drug- or IPC-treated groups were the result of their direct or indirect effects on other determinants of IS, such as cell signaling pathways. Six animals were excluded from data analysis due to a high collateral blood flow (endocardial, >0.15 ml·min−1·g−1).

Hemodynamics.

Heart rate and mean arterial blood pressure at baseline between groups or at 30 min after occlusion or 3 h after reperfusion were not significantly different (Table 2). These data further suggest that changes in IS were not the result of changes in peripheral hemodynamics.

Effects of 11,12- and 14,15-EET on myocardial IS.

IS/AAR in the four treatment groups was significantly reduced compared with that in the control group (Fig. 2A). 11,12-EET at 0.128 mg/kg markedly reduced IS/AAR (6.4 ± 1.9 and 8.8 ± 2.1%, respectively) compared with that in the control group (22.1 ± 1.8%), whether administered 15 min before occlusion or 5 min before reperfusion. Similarly, 14,15-EET at 0.128 mg/kg also produced a marked reduction in IS/AAR (8.4 ± 2.4% and 9.7 ± 1.4%, respectively). Exogenous administration of the major metabolite of 14,15-HETE, 14,15-DHET, had no effect on IS/AAR (Fig. 2A). The beneficial effect of 11,12-EET pretreatment on IS/AAR (6.4 ± 1.9%) was completely abolished by pretreatment with the KATP channel blocker glibenclamide (24.3 ± 4.6%, Fig. 2B).

Effects of MS-PPOH on IPC- and DDMS-induced cardioprotection.

A brief 5-min period of IPC of the heart 10 min before the 60-min occlusion decreased IS/AAR to 8.7 ± 1.8%, similar to previous results obtained in our laboratory (10). Pretreatment with MS-PPOH did not block the effects of IPC to reduce IS/AAR (10.5 ± 2.0%, Fig. 3A). In contrast, DDMS produced a marked reduction in IS/AAR (4.9 ± 2.9%, Fig. 3B), similar to that previously observed in our laboratory (10); however, the protective effects of DDMS were completely abolished by pretreatment with MS-PPOH (22.2 ± 3.4%, Fig. 3B).

A: IS/AAR (in %) for the following groups: control, IPC, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) (CYP epoxygenase inhibitor). AAR/LV was not significantly different among groups (data not shown). IPC produced significant reduction in IS/AAR, and this effect was not blocked by pretreatment with MS-PPOH (0.646 mg/kg). B: IS/AAR for the following groups: control; N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a CYP ω-hydroxylase inhibitor ( 0.81 mg/kg); MS-PPOH alone (0.646 mg/kg); or MS-PPOH + DDMS. AAR/LV (in %) was not significantly different among groups (data not shown). DDMS produced a marked reduction in IS/AAR, and this effect was completely antagonized by the CYP epoxygenase inhibitor MS-PPOH at a dose that had no effect on IS/AAR when administered by itself. Values are means ± SE; n = 8 dogs/group. *P < 0.01, significantly less than control.

Plasma CYP metabolites of AA, 14,15-EET, and 14,15-DHET.

When dogs were pretreated with 14,15-EET, there was an increase from nondetectable concentrations in coronary venous blood to a peak of 0.5 ng/ml immediately after drug injection, which remained at a concentration between 0.25 and 0.5 ng/ml throughout occlusion and reperfusion (Fig. 4B). 14,15-DHET, the metabolite of 14,15-EET, also increased to a peak of ∼5 ng/ml, which remained at that level throughout occlusion and fell during the 3 h of reperfusion to ∼1 to 2 ng/ml at 3 h. Infusion of 14,15-DHET at a dose calculated to approximate the concentration of 14,15-DHET, obtained with the injection of 14,15-EET, peaked at ∼4.5 ng/ml and remained at this concentration throughout reperfusion (Fig. 4A). These data suggest that the protective effect of 14,15-EET is not due to increases in its metabolite 14,15-DHET.

Plasma concentrations of 14,15-DHET after administration of vehicle, 14,15-EET before ischemia, and those of 14,15-DHET after administration of this same compound at 0.155 mg/kg before ischemia. There were no detectable concentrations of 14,15-DHET in vehicle-treated animals; however, both 14,15-EET and 14,15-DHET produced nearly equivalent increases in plasma 14,15-DHET at least until 30 min of reperfusion (A). At 1 and 3 h of reperfusion, EETs were decreased to concentrations below the group that had 14,15-DHET administered. B: 14,15-EET concentrations in vehicle-treated group and group given only 14,15-EET. There were no detectable EET concentrations released in vehicle-treated dogs; however, there was a significant increase in 14,15-EETs at 30 min after EET administration, which remained elevated throughout reperfusion (R). O, occlusion.

DISCUSSION

We have previously demonstrated that CYP ω-hydroxylases and their AA metabolite 20-HETE have a detrimental role in enhancing myocardial ischemia-reperfusion injury in the canine myocardium (10, 12). In the present study, we demonstrate that exogenous administration of two CYP epoxygenase products, 11,12-EET and 14,15-EET, markedly reduce IS in canine hearts subjected to a 60-min occlusion period followed by 3 h of reperfusion. The reduction of IS was observed whether the EET was administered 15 min before occlusion or at 5 min before reperfusion. This latter observation, which suggests that the EETs may reduce reperfusion injury, has important clinical implications because most patients suffering an acute ischemic event would be administered cardioprotective therapies after a certain period of occlusion and not before the coronary occlusion. Of additional importance, the magnitude of the reduction in IS/AAR after EET administration was equivalent to that of IPC, thought to be the most powerful cardioprotective intervention thus far demonstrated. Similar to IPC (5), the protective effects of the EETs are at least partially the result of opening of KATP channels, although the results with glibenclamide do not discern between the importance of the sarc- or mitoKATP channel. Taken together, these results suggest that activation of CYP ω-hydroxylases and administration of exogenous 20-HETE have significant detrimental effects on the canine myocardium during ischemia and/or after reperfusion and that exogenous administration of two regioisomers of the CYP epoxygenase pathway, 11,12- and 14,15-EET, demonstrated potent cardioprotective effects in canine hearts. Because the protective effects of the EETs and IPC were nearly equally effective, we initially hypothesized that the effects of IPC may be the result of an increase in EET synthesis; however, our results with the CYP epoxygenase inhibitor MS-PPOH suggests that this is not likely. MS-PPOH did not block the protective effect of IPC, whereas the effect of a CYP ω-hydroxylase inhibitor DDMS, which we (10) had previously shown to also be markedly cardioprotective in dogs, presumably by reducing the production of 20-HETE and shifting the endogenous balance in favor of EET production, was completely blocked by MS-PPOH. The additive effects of DDMS and IPC in our previous work also support the hypothesis that IPC and inhibition of the CYP ω-hydroxylase pathway each have distinct signaling pathways, although they may converge on the KATP channel downstream.

Several recent studies (13, 15) indicate that CYP epoxygenases and their AA metabolites, the EETs, are cardioprotective in mice. In plasma samples, obtained and published in several recent references from our laboratory (10, 12), only 11,12- and 14,15-DHET but not the parent compounds were detected, suggesting the possibility that the EETs were rapidly hydrolyzed by soluble epoxide hydrolase (sEH). Despite their rapid hydrolysis and avid protein binding, the cardioprotective effect of the exogenous EETs appears to be very long lasting, which suggests that like IPC, their effect turns on a signaling pathway that is not immediately inactivated. Nevertheless, the synthesis of new EET analogs that are resistant to breakdown by sEH may be desirable for future clinical applications. In this regard, the effect of several sEH inhibitors has recently been suggested to have protective effects as hypotensive and lipid-lowering agents and may also be expected to have cardioprotective effects by enhancing or preserving EET concentrations in the ischemic and/or reperfused myocardium (14).

Interestingly, the protective effect of the EETs was completely blocked by the KATP channel blocker glibenclamide. This finding in dogs was not that surprising because the EETs have been previously shown to activate sarcKATP channels in membrane patches (6, 7) isolated from rat hearts and mitoKATP channels as assessed by changes in flavoprotein fluorescence in mitochondria isolated from mouse hearts (13). Further studies are necessary to determine the relative importance and timing of opening of these two channels in dogs or other species.

In conclusion, the present results indicate that exogenous administration of two of the major regioisomers of the CYP epoxygenase pathway, 11,12-EET and 14,15-EET, both produce equivalent and markedly significant reductions in IS/AAR in the canine heart, whether given before occlusion or at reperfusion, and that these effects are mediated by opening of cardiac KATP channels. The cardioprotective effects observed were nearly equivalent to that of IPC, although the present results using the CYP epoxygenase inhibitor MS-PPOH suggest that these two interventions may have some fundamental differences in their signaling pathways. Although the cellular mechanism(s) responsible for the protection observed after EET administration are yet to be determined, these data suggest potential therapeutic targets for intervention in myocardial ischemia-reperfusion injury. Blockade of the CYP ω-hydroxylase pathway by agents such as DDMS or activation of the CYP epoxygenase pathway and subsequent increase in EET formation may be two interrelated mechanisms, which, when optimally combined, may result in a powerful novel approach for combating ischemic heart disease.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-74314-01.

Footnotes

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